PAL is a non-mammalian enzyme widely distributed in plants (Koukol, et al., J. Biol. Chem., 236, pp. 2692-2698 (1961); Hanson, et al., The Enzymes (Boyer, P. D., Ed.), Academic Press, New York, Vol. 7, pp. 75-166 (1972); Poppe, et al., (2003) ibid.) some fungi (Rao, et al., Can. J. Biochem., 4512), pp. 1863-1872 (1967)) and yeast (Abell, et al., Methods Enzymol. 142, pp. 242-253 (1987)) and can also be recombinantly produced in Escherichia coli. 
A representative list of PALs includes: Q9ATN7 Agastache rugosa; O93967 Amanita muscaria (Fly agaric); P35510, P45724, P45725, Q9SS45, Q8RWP4 Arabidopsis thaliana (Mouse-ear cress); Q6ST23 Bambusa oldhamii (Giant timber bamboo); Q42609 Bromheadia finlaysoniana (Orchid); P45726 Camellia sinensis (Tea); Q9MAX1 Catharanthus roseus (Rosy periwinkle) (Madagascar periwinkle); Q9SMK9 Cicer arietinum (Chickpea); Q9XFX5, Q9XFX6 Citrus clementina x Citrus reticulate; Q42667 Citrus limon (Lemon); Q8H6V9, Q8H6W0 Coffea canephora (Robusta coffee); Q852S1 Daucus carota (Carrot); O23924) Digitalis lanata (Foxglove); O23865) Daucus carota (Carrot); P27991) Glycine max (Soybean); O04058) Helianthus annuus (Common sunflower); P14166, (Q42858) Ipomoea batatas (Sweet potato); Q8GZR8, Q8W2E4 Lactuca sativa (Garden lettuce); O49835, O49836 Lithospermum erythrorhizon; P35511, P26600 Lycopersicon esculentum (Tomato); P35512 Malus domestica (Apple) (Malus sylvestris); Q94C45, Q94F89 Manihot esculenta (Cassaya) (Manioc); P27990 Medicago sativa (Alfalfa); P25872, P35513, P45733 Nicotiana tabacum (Common tobacco); Q6T1C9 Quercus suber (Cork oak); P14717, P53443, Q7M1Q5, Q84VE0, Q84VE0 Oryza sativa (Rice); P45727 Persea americana (Avocado); Q9AXI5 Pharbitis nil (Violet) (Japanese morning glory); P52777 Pinus taeda (Loblolly pine); Q01861, Q04593 Pisum sativum (Garden pea); P24481, P45728, P45729 Petroselinum crispum (Parsley) (Petroselinum hortense); Q84LI2 Phalaenopsis x Doritaenopsis hybrid cultivar; P07218, P19142, P19143 Phaseolus vulgaris (Kidney bean) (French bean); Q7XJC3, Q7XJC4 Pinus pinaster (Maritime pine); Q6UD65 Populus balsamifera subsp. trichocarpa x Populus deltoides; P45731, Q43052, O24266 Populus kitakamiensis (Aspen); Q8H6V5, Q8H6V6 Populus tremuloides (Quaking aspen); P45730 Populus trichocarpa (Western balsam poplar); O64963 Prunus avium (Cherry); Q94ENO Rehmannia glutinosa; P11544 Rhodosporidium toruloides (Yeast) (Rhodotorula gracilis); P10248 Rhodotorula rubra (Yeast) (Rhodotorula mucilaginosa); Q9M568, Q9M567 Rubus idaeus (Raspberry); P31425, P31426 Solanum tuberosum (Potato); Q6SPE8 Stellaria longipes (Longstalk starwort); P45732 Stylosanthes humilis (Townsville stylo); P45734 Trifolium subterraneum (Subterranean clover); Q43210, Q43664 Triticum aestivum (Wheat); Q96V77 Ustilago maydis (Smut fungus); P45735 Vitis vinifera (Grape); and Q8VXG7 Zea mays (Maize).
Histidine ammonia-lyase (HAL, E.C. 4.3.1.3) is found in mammalian as well as bacterial sources (Taylor, et al., J. Biol. Chem., 265(30), pp. 18192-18199 (1990); Suchi, et al., Biochim. Biophys. Acta, 1216(2), pp. 293-295 (1993)) and the crystal structure of histidase from Pseudomonas putida is known (Schwede, et al., Biochemistry, 38(17), pp. 5355-5361 (1999)). HAL from Corynebacteriaceae has been proposed to be used for combination therapy with L-histidinol to treat histidine- and/or histamine-dependent pathologies such as human respiratory syncytial virus (HSV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), and cancer (U.S. Patent Application No. 20020052038).
A representative list of HALs includes: Q9 KWE4 (Agrobacterium rhizogenes), Q8U8Z7 (Agrobacterium tumefaciens), Q6 KPK9, Q81Y45 (Bacillus anthracis), Q733H8, Q81AC6 (Bacillus cereus), Q9 KBE6 (Bacillus halodurans), P10944 Bacillus subtilis), Q8A4B3 (Bacteroides thetaiotaomicron), Q8G4X5 (Bifidobacterium longum), Q89GV3 (Bradyrhizobium japonicum), Q8YD10 (Brucella melitensis), Q8FVB4 (Brucella suis), Q20502 (Caenorhabditis elegans), P58082 (Caulobacter crescentus), Q7P188 (Chromobacterium violaceum), Q891Q1 (Clostridium tetani), Q9RZ06 (Deinococcus radiodurans), Q8RFC2, Q8RDU4, Q7P5N4 (Fusobacterium nucleatum), Q7NCB3 (Gloeobacter violaceus), Q9HQD5 (Halobacterium sp.), P42357 (Homo sapiens (Human)), P35492 (Mus musculus (Mouse)), Q7N296 (Photorhabdus luminescens), Q6L2V9 (Picrophilus torridus), Q7MX86 (Porphyromonas gingivalis), Q9HU85 (Pseudomonas aeruginosa), Q9HU90 (histidine/phenylalanine ammonia-lyase, Pseudomonas aeruginosa), Q8VMR3 (Pseudomonas fluorescens), Q88CZ7, P21310 (Pseudomonas putida), Q87UM1, Q87UM2, Q87V42 (Pseudomonas syringae), Q8XW29 (Ralstonia solanacearum, Pseudomonas solanacearum), P21213 (Rattus norvegicus (Rat), Q98310, Q987B4, Q98JY1, Q98NG3 (Rhizobium loti, Mesorhizobium loti), 031197 Rhizobium meliloti (Sinorhizobium meliloti), Q8Z896 (Salmonella typhi), Q8ZQQ9 (Salmonella typhimurium), Q8E9B0, Q8EKJ4 (Shewanella oneidensis), Q99XG3, Q8NYY3 (Staphylococcus aureus), Q93TX3 (Stigmatella aurantiaca), Q9EWW1 (Streptomyces coelicolor), P24221 (Streptomyces griseus), Q8K5L, Q8NZ46, P58083 (Streptococcus pyrogenes), Q9HLI6 (Thermplasma acidophilum), Q8RBH4 (Thermoanaerobacter tengcongensis), Q978N8 (Thermoplasma volcanium), Q73Q56 (Treponema denticola), Q9KSQ4 (Vibrio cholerae), Q87Q77 (Vibrio parahaemolyticus), Q8DA21, Q7MK58, Q7MMJ6, Q8DFZ8 (Vibrio vulnificus), Q8PLZ8 (Xanthomonas axonopodis), Q8PAA7 (Xanthomonas campestris), Q8ZA10 (Yersinia pestis).
Enzyme Substitution Therapy for PKU Treatment
Numerous studies have focused on the application of the enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) for enzyme substitution treatment of PKU (Hoskins, Lancet, i(8165), pp. 392-394 (1980); Gilbert, et al., Biochem. J., 199(3):715-723 (1981); Hoskins, et al., (1982) ibid.; Sarkissian, et al., (1999) ibid.; Liu, et al., Artif. Cells Blood Substit. Immobil. Biotechnol., 30(4):243-257 (2002); Wieder, J Biol Chem., 254(24):12579-12587 (1979); Gamez, In press; Ambrus, et al., J. Pharmacol. Exp. Ther., 224(3):598-602 (1983); Ambrus, et al., Science, 201(4358):837-839 (1978); Kalghatgi, Res. Commun. Chem. Pathol. Pharmacol., 27(3):551-561 (1980); Ambrus, Res. Commun. Chem. Pathol. Pharmacol., 37(1):105-111 (1982); Gilbert, et al., Biochem. Biophys. Res. Commun., 131(2):557-563 (1985); Pedersen, Res. Commun. Chem. Pathol. Pharmacol., 20(3):559-569 (1978); Marconi, et al., Biochimie, 62(8-9):575-580 (1980); Larue, et al., Dev. Pharmacol. Ther., 9(2):73-81 (1986); Ambrus, C. M., et al., (1987) ibid.; Bourget, et al., Appl. Biochem. Biotechnol., 10:57-59 (1984); Bourget, et al., FEBS Lett., 180(1):5-8 (1985); Bourget, et al., Biochim. Biophys. Acta, 883(3):432-438 (1986); Chang, et al., Artif. Cells Blood Substit. Immobil. Biotechnol., 23(1):1-21 (1995); Chang, et al., Mol Biotechnol., 17(3):249-260 (2001); U.S. Pat. No. 5,753,487).
Phenylketonuria (PKU) is an inborn error of amino acid metabolism that results from impaired activity of hepatic phenylalanine hydroxylase (PAH), the enzyme responsible for the metabolism of phenylalanine. Patients with PAH mutations that lead to PKU and hyperphenylalaninemia (HPA) display impaired neurophysiological functioning and reduced cognitive development. For patients that have severe PKU, there is the potential for irreversible mental retardation unless phenylalanine is controlled at low levels using dietary restrictions. PAL converts phenylalanine to ammonia and trans-cinnamic acid, a harmless metabolite, which is further metabolized and excreted in the urine as hippurate ((Hoskins, et al., (1980) ibid; Hoskins, J. A., et al., “The metabolism of cinnamic acid by healthy and phenylketonuric adults: a kinetic study”, Biomed Mass Spectrom, 11(6), pp. 296-300 (1984)).
Current treatment for PKU involves the adherence to a restricted diet for life that is low in proteins and the amino acid phenylalanine (Levy, Proc. Natl. Acad. Sci. U.S.A., 96(5), pp. 1811-1813 (1999)). This dietary therapy is difficult to maintain (Matalon, et al., Genet. Med., 6(1), pp. 27-32 (2004); Woolf, et al., Arch. Dis. Child., 33(167), pp. 31-45 (1958); Kim, Mol Ther., 10(2), pp. 220-224 (2004)) and does not always eliminate the damaging neurological effects that can be caused by elevated phenylalanine levels (Sarkissian, et al., Mol. Genet. Metab., 69, pp. 188-194 (2000)); less than ideal dietary control during pregnancy can lead to birth defects (Levy, H. L., (1999) ibid.). In addition, it is very difficult for PKU/HPA patients to live a normal life while following the restrictive diet, and the dietary therapy can be associated with deficiencies of several nutrients, some of which are detrimental for brain development (Levy, H. L., (1999) ibid.). Most low phenylalanine diet products have organoleptic properties sufficiently unsatisfactory that compliance with this treatment is compromised (Levy, H. L., (1999) ibid.). Therefore, development of a therapeutic treatment would assist the current dietary treatment and prevent the neurological damages inflicted on those individuals with PKU, particularly for those patients with the most severe forms of the disease.
In 1999, Scriver and colleagues reported their initial studies on the use of the enzyme PAL from Rhodosporidium toruloides (Sarkissian, C. N., et al., 1999 ibid.) for PKU enzyme substitution applications. Mouse PKU and HPA model studies demonstrated that PAL administration (either by i.p. injection or orally using either PAL in combination with aprotinin protease inhibitor or PAL recombinantly expressed and present inside E. coli cells) was able to successfully lower blood plasma phenylalanine levels. In addition, preliminary studies describing the use of PAL with PKU patients have shown reduction in phenylalanine levels using PAL administered in enteric-coated gelatin capsules (Hoskins, J. A., et al., (1980) ibid.) or using an extracorporeal enzyme factory (Ambrus, et al., Ann. Intern. Med., 106(4), pp. 531-537 (1987)). However, the sensitivity of PAL to protease inactivation (low activity in gastric conditions due to protease degradation) and the reduced half-life found after repeated in vivo injection (due to elicitation of an immune response) limits further development of the native PAL protein as a clinical therapeutic.
Other Therapeutic Uses
The use of PAL for cancer treatment has also been suggested based on its ability to limit the nutrient supply of phenylalanine to cancer cells and thereby inhibit neoplastic growth (Fritz, et al., J Biol Chem. 251(15):726 (1976); Roberts, et al., Cancer Treat Rep., 60(3):261-263 (1976); Shen, et al., Cancer Res. 37(4):1051-1056 (1977); Shen, et al., Cancer Treat Rep. 63(6):1063-1068 (1979); Wieder, et al, J Biol Chem., 254(24):12579-12587 (1979)). However, intravenously injected pegylated PAL was cleared rapidly from circulating blood after the 13th injection. In addition, PAL-mediated reduction in phenylalanine prevented the proliferation of murine leukemia and metastatic melanoma (Abell, et al., Cancer Res. 33:2529-2532 (1973)), Roberts, et al., ((1976) ibid) Shen, et al., ((1977) ibid)).
PAL has also been used for tyrosinemia (Marconi, W., et al., (1980) ibid.). Histidine ammonia-lyase (HAL) has been used in enzyme substitution therapy for histidinemia treatment. Histidinemia is an autosomal recessive disorder of histidine metabolism due to defective HAL, and is traditionally a benign condition (Taylor, R. G., et al., (1990) ibid.).
Additional Uses of PAL
PAL has an important industrial use for the synthesis of L-phenylalanine methyl ester (for Aspartame production (D'Cunha, et al., Enzyme and Microbial Technology, 19(6), pp. 421-427 (1996); Hamilton, et al., Trends in Biotechnol., 3(3), pp. 64-68 (1985)) and other substituted L-phenylalanine derivatives that are used as pharmaceutical precursors (U.S. Patent App. 20020102712).
PAL also has agricultural importance, being the initial enzymatic process leading to the phenylpropaniods that produce lignins, coumarins, and flavaniods in plants, fungi, and bacteria. All phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) produce cinnamic acid, which is a precursor for lignins, flavonoids, and coumarins in plants (Alunni, et al., Arch Biochem Biophys., 412(2), pp. 170-175 (2003)). Hence, modulation of PAL activity can influence a number of agricultural phenomena such as the browning of fruit. In addition, structure-based drug design of active site PAL inhibitors could lead to effective herbicides (Poppe, L., et al., ibid.).
Although PAL potentially has various industrial and therapeutic applications, the use of PAL may be limited by reduced specific activity and proteolytic instability. Similar to other therapeutic proteins, use of PAL as an enzyme therapy is accompanied by several disadvantages such as immunogenicity and proteolytic sensitivity. Further, a delicate balance is required between substrate affinity and enzyme activity to achieve and maintain control of plasma phenylalanine levels within a normal somewhat narrow range in disorders characterized by hyperphenylalanemia. As yet, a concerted effort toward improving these parameters has not been made due to a paucity of structural and biochemical knowledge regarding this protein.
Protein Therapeutics and Effective Redesign for Therapeutic Advantage
Numerous proteins are used therapeutically to alleviate metabolic deficiencies caused by genetic disorders. Among the most notable examples are parenterally administered insulin for the treatment of diabetes, alpha-glucosidase for enzyme replacement therapy in Pompe's disease (and other enzymes that are used for other lysosomal storage diseases (“Enzyme Therapy In Genetic Diseases”, Birth Defects Original Article Series, Volume 9, E. D. Bergsma, Ed. Baltimore: Williams and Wilkins Company (1973))), interferon-alpha for hepatitis C or cancer treatment, and adenosine deaminase for severe combined immunodeficiency (SCID) therapy (Russell, et al., Clin. Genet., 55(6): pp. 389-394 (1999)). Unfortunately, the lifetimes of these injected foreign proteins are usually diminished due to acute allergic reactions and rapid clearance from the bloodstream.
The efficacy of protein therapeutics can be improved with protein engineering methods, including rational design, directed evolution, chemical modification, and combinatorial optimization strategies. Rational design requires the availability of three-dimensional structural information and consideration of side-chain orientations and mobilities in the context of the structure, combined with generic properties such as side-chain hydrophobicity, polarity, charge, electronic contributions, and propensities to form specific secondary structures. Among rational protein modification methods, a most promising solution involves producing variants using structure-based protein engineering.
Numerous examples of structure-based protein engineering exist, wherein improved properties have been designed into proteins using structure-based design techniques (Lazar, et al., Curr. Opin. Struct. Biol., 13(4), pp. 513-518 (2003); Marshall, et al., Drug Discov. Today, 8(5), pp. 212-221 (2003)). Site-directed mutagenesis of proteins can be used to generate protein variants containing truncations, insertions, and/or point mutations, leading to improved stability, activity, and/or altered activity (Brannigan, et al., Nat Rev Mol Cell Biol., 3(12), pp. 964-970 (2002)). Chimeras, or combinations of two proteins, have been successfully exploited for a number of therapeutic antibody and protein examples. Additional effective protein engineering approaches have used specific protein loop re-engineering (Chen, et al., Proc. Natl. Acad. Sci. U.S.A., 90(12), pp. 5618-5622 (1993)), loop swapping (Wilks, et al., Biochemistry, 31(34), pp. 7802-7806 (1992); 36 Hedstrom, et al., Science, 255(5049), pp. 1249-1253 (1992)), and subdomain shuffling (Hopfner, et al., Proc. Natl. Acad. Sci., U.S.A., 95(17), pp. 9813-9818 (1998)) approaches.
Immunologic Response Reduction for Protein Therapeutics
Numerous strategies have been devised for minimizing immune responses of therapeutically-administered proteins (Chirino, et al., Drug Discov. Today, 9(2), pp. 82-90 (2004); U.S. Pat. Nos. 6,686,164 and 6,461,849). For some protein classes such as antibodies, increasing human sequence content (chimeras and/or ‘humanization’) has reduced immunoreactivity, whereas another effective strategy wherein protein solution properties have been improved (e.g. to reduce aggregation propensity) has also led to reduced immune response. Numerous proteins have had antibody epitopes and agretopes removed using iterative site-directed mutagenesis including replacement of hydrophobic and charged residues with polar neutral residues, alanines, or computationally-selected residues to produce less immunoreactive protein variants. In one example, site-directed mutagenesis of streptavidin was used to reduce immunogenicity (Meyer, et al., Protein Sci., 10(3), pp. 491-503 (2001)). In this case, surface “veneering” was used to mutate surface residues capable of forming high energy ionic or hydrophobic interactions to remove such potential interacting sites. Mutants producing high yields of active tetrameric protein were then tested for reduced antibody recognition in mice and humans, and minimized antibody response upon mutant injection in rabbits. In general, substitution of smaller neutral residues for charged, aromatic, or large hydrophobic surface residues reduced the ability to elicit an immune response in rabbits.
Site-directed mutagenesis has been successfully used for point mutagenesis to alleviate immunoreactivity. For example, site-directed rational modification of antigenic determinants was used to downregulate the CD8(+) and CD4(+) T lymphocyte responses (Abrams, et al. Curr. Opin. Immunol., 12(1), pp. 85-91 (2000)). Alternate immunoreactivity reduction routes include the removal of agretopes, the generation of variants less susceptible to antigen-processing cell recognition or binding, or the reduction or removal of MHC binding ability. Different approaches have relied upon humanization (making protein variants with more human sequence content), surface veneering (making protein variants with less immunoreactive surface features, and other similar methods wherein specific mutations are made and then screened for lessened immunoreactivity. For example, the “Immunostealth” method uses a combination of in silico sequence analysis methods and high-throughput in vitro biochemical screening assays to identify HTL epitopes, followed by rational modification to alter their HTL binding capacity, resulting in protein variants non-recognizable by the immune system (Tangri, et al., Curr. Med. Chem., 9(24), pp. 2191-2199 (2002)). In addition, the methods of structure-based protein engineering can be combined with more ‘random’ protein modification methods, such as directed evolution, that can use screening and/or selection to develop more favorable protein variants.
In another approach, the chemical conjugation of a water-soluble polymer, such as polyethylene glycol (PEG), to the protein of interest is a common approach often used to extend the half-lives of proteins in vivo. Generally, polyethylene glycol molecules are connected to the protein via a reactive group found on the protein. Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such attachment. The covalent coupling of activated PEG molecules to the protein of interest (pegylation) has been shown to increase circulation half-times, reduce immunogenicity and antigenicity, and allow retention of bioactivity (Harris, et al., Nat. Rev. Drug Discov., 2(3), pp. 214-221 (2003); Greenwald, et al., Adv Drug Deliv Rev., 55(2), pp. 217-250 (2003); Veronese, et al., Adv Drug Deliv Rev., 54(4), pp. 453-456 (2002); Mehvar, J. Pharm. Pharmaceut. Sci., 3(1), p. 125-136 (2000); Delgado, et al., Critical Reviews in Therapeutic Drug Carrier Systems, 9(3-4), pp. 249-304 (1992)).
Currently, a number of pegylated protein therapeutics have received FDA approval and are being used parenterally to treat a number of diseases such as: hepatitis C and metastatic renal cell carcinoma using PEG-INTRON™ (pegylated interferon-alpha2b [Wang, et al., Adv Drug Deliv Rev., 54(4), pp. 547-570 (2002)]) or PEGASYS™ (pegylated interferon-alpha2a [Rajender Reddy, et al., Adv Drug Deliv Rev., 54(4), pp. 571-586 (2002)]); acute lymphoblastic leukemia using ONCASPAR™ (PEGASPARGASE™ or pegylated L-asparaginase [Graham, Adv Drug Deliv Rev., 55(10), pp. 1293-1302 (2003)]); severe combined immunodeficiency (SCID) using ADAGEN™ (bovine PEGADEMASE™ or pegylated adenosine deaminase [Hershfield, Immunodeficiency, 4(1-4), pp. 93-97 (1993)]); and acromegaly using SOMAVERT™ (PEGVISOMANT™ or pegylated human growth hormone antagonist [Parkinson, et al., Adv Drug Deliv Rev., 55(10), pp. 1303-1314 (2003)]).
Pegylated Proteins
In addition to the pegylation of native protein, site-specific pegylation of proteins has also been performed. Examples of site-specific pegylation include Cys-pegylated IL-3 (U.S. Pat. No. 5,166,322 and WO 90/12874) and Cys-pegylated IL-2 (U.S. Pat. No. 5,206,344) as well as Lys-pegylated purine nucleoside phosphorylase (Hershfield, et al., Proc. Natl. Acad. Sci. USA, 88, pp. 7185-7189 (1991)), an N-terminal selectively pegylated lysine-deficient mutant TNF-α (Yoshioka, et al., Biochem Biophys Res Commun., 315(4), pp. 808-814 (2004)), an N-terminally site-specific pegylated G-CSF (Kinstler, et al., Pharm. Res., 13(7), pp. 996-1002 (1996)), and gylcosylation site-specific pegylated IL-2 (Goodson, et al., Biotechnology, 8(4), pp. 343-346 (1990)). U.S. Pat. No. 6,451,986 discloses site-specific mutation and pegylation of p75 tumor necrosis factor receptor, and U.S. Pat. No. 5,766,897 discloses site-specific pegylation via an exisiting cysteine residue or introduction of a site-specific cysteine residue (at an N-linked glycosylation site or the position of a residue that is normally solvent-accessible in the naturally-occurring protein). Hermeling, S., et al. discuss the use of site-specific PEG attachment around possible antigenic epitope regions to reduce immunogenicity (Hermeling et al. Pharm Res., 21(6), pp. 897-903 (2004)). Similar approaches to improve protein in vivo lifetimes have been adopted for a Cys-pegylated humanized anti-interleukin-8 antibody (Leong, et al., (2001) ibid.) and an N-terminal aldehyde activated alpha-amine group derivatized on epidermal growth factor (Lee, et al., Pharm. Res., 20(5), pp. 818-825 (2003)). In a related approach, a hyper-glycosylated form of human erythropoietin displayed an improved serum half-life and greater in vivo potency, thereby allowing for less frequent administration to obtain the same biological response (Egrie, et al., Br J Cancer, 84(Suppl 1), pp. 3-10 (2001)); chemical modification of this form of erythropoietin improved in vivo efficacy even more (U.S. Pat. No. 6,586,398). In one example, pegylation plus additional site-directed mutagenesis was necessary in order to engineer an active IL-15 variant (Pettit, et al., J. Biol. Chem., 272(4), pp. 2312-2318 (1997)).
Native Rhodotorula glutinis PAL stabilized with pegylation has been investigated as a therapeutic agent in cancer therapy. However, these agents exhibited residual immunogenicity and protease sensitivity, thereby precluding them from use in human, such as in clinical testing (Wieder, J Biol Chem., 254(24), pp. 12579-12587 (1979)).
The pegylation of PAL to improve L-phenylalanine synthetic capability for industrial applications, and to reduce the immunogenicity of PAL for therapeutic applications has been reported. U.S. Pat. Nos. 4,562,151 and 5,981,239 disclose the use of polyethylene glycol as an agent to improve the activity of PAL for the production of L-phenylalanine and L-phenylalanine analogs, respectively. U.S. patent application Nos. 20020102712 and 20030082238 describe the use of PEG to stabilize, solubilize, and/or reduce the immunogenicity of PAL for phenylketonuria treatment. U.S. Pat. No. 5,766,897 discloses the use of site-specific introduction of cysteine residues in proteins such as PAL for covalent PEG attachment to improve half-life, decrease immunogenicity and antigenicity, and retain substantially the same level of biological activity.
Oral Therapeutics
Parenterally administered protein therapeutics have demonstrated their clinical effectiveness, but alternative routes of administration are also used. For example, there are two “over-the-counter” oral enzyme replacement therapies that are used for dietary digestion remediation. BEANO™, alpha-galactosidase from Aspergillis niger (a food grade mold), is used to correct digestive deficiencies associated with deficient carbohydrate processing (and intestinal gas formation) from legume consumption. Additionally, LACTAID™, an orally active form of the lactase enzyme, is used to alleviate problems associated with lactose (milk sugar) intolerance. Both these products demonstrate that orally administered enzymes can function in the gastrointestinal tract and can successfully correct dietary metabolic deficiencies.
Native R. toruloides PAL is very susceptible to protease inactivation (Sarkissian, C. N., et al., (1999) ibid.), requiring either site-directed mutagenesis and/or the introduction of protein surface protective features such as pegylation to produce an orally-effective PAL variant. Additional protease protection can be provided for by using microcapsules (Wang, et al., “Biomater. Artif. Cells Immobilization Biotechnol., 21(5), pp. 637-646 (1993)). Complex microcapsules could be used as an additional measure to protect a therapeutic enzyme from inactivation in both the stomach and the intestine. Semi-permeable microcapsules can be further encapsulated by enteric-soluble materials to protect the microcapsules from gastric juice. When the encapsulated enzyme passes into the intestine, the small molecule L-phenylalanine can rapidly diffuse and equilibrate across the semipermeable membrane, allowing conversion to non-toxic products via the encapsulated enzyme.
U.S. Pat. No. 5,753,487 discloses mutants of R. toruloides PAL wherein one or more amino acids susceptible to proteolytic cleavage are replaced by other amino acids less susceptible to proteolytic cleavage.
Thus, there remains a need for PAL and HAL molecules with optimal kinetic characteristics including potent catalytic activity and greater biological half-life, greater biochemical stability and attenuated immunogenicity.